A biochemical analysis into the co-translational folding of a G protein-coupled receptor; GPR35

The folding and targeting of membrane proteins poses a major challenge to the cell, as they must remain insertion competent while their highly hydrophobic transmembrane (TM) domains are transferred from the ribosome, through the aqueous cytosol and into the lipid bilayer. The biogenesis of a mature membrane protein takes place through the insertion and integration into the lipid bilayer. A number of TM proteins have been shown to gain some degree of secondary structure within the ribosome tunnel and to retain this conformation throughout maturation. Although studies into the folding and targeting of a number of membrane proteins have been carried out to date, there is little information on one of the largest class of eukaryotic membrane proteins; the G-protein-coupled receptors (GPCRs). This project studies the early folding events of the human ortholog of GPR35. To analyse the structure of the 1st TM domain, intermediates were generated and assessed by the biochemical method of pegylation (PEG-MAL). A structurally-similar microbial opsin (Bacterioopsin) was also used to investigate the differences in the early protein folding within eukaryotic and prokaryotic translation systems. Results showed that neither the 1st TM domain of GPR35 nor Bacterioopsin were capable of compacting in the ribosome tunnel before their N-terminus reached the ribosome exit point. The results for this assay remained consistent whether the proteins were translated in a eukaryotic or prokaryotic translation system. To examine the communication mechanism between the ribosome, the nascent chain and the protein targeting pathway, crosslinking experiments were carried out using the homobifunctional lysine cross-linker BS3. Specifically, the data generated here show that the nascent chain of GPR35 reaches the ribosomal protein uL23 in an extended conformation and interacts with the SRP protein as it exits the ribosome tunnel. This confirms the role of SRP in the co-translational targeting of GPR35. Using these methods insights into the early folding of GPCRs has been obtained. Further experiments using site-directed mutagenesis to reduce hydrophobicity in the 1st TM domain of GPR35, highlighted the mechanisms by which GPCRs are targeted to the endoplasmic reticulum. Confirming that hydrophobicity within the signal anchor sequence is essential of SRP-dependent targeting. Following the successful interaction of the nascent GPR35 and SRP, GPR35 is successfully targeted to ER membranes, shown here as dog pancreas microsomes (DPMs). Glycosylation of the GPR35 N-terminus was used to determine nascent chain structure as it is inserted into the ER membrane. These glycosylation experiments confirm that TM1 has obtained its compacted state whilst residing in the translocon. Finally, a site-specific cross-linking approach using the homobifunctional cysteine cross-linker, BMH, was used to study the lateral integration of GPR35 into the ER. Cross-linking of GPR35 TM1 and TM2 could be detected adjacent to a protein of ~45kDa, believed to be Sec61α. The loss of this adduct, as the nascent chain extends, showed the lateral movement of GPR35 TM1 from the translocon was dependent on the subsequent synthesis of TM2.

CIBERESPACIO: REINVENTANDO LA METÁFORA DE LO HUMANO

La pregunta sobre el humanismo está íntimamente ligada con las metáforas que las distintas sociedades en diferentes momentos han utilizado no sólo para comprender las relaciones con su entorno, sino también para estructurar su visión sobre sí mismas.En la Edad Media, el topos del microcosmos y el macrocosmos no sólo servía para reconocer en el mundo los principios de la analogía y la similitud como articuladores de las «realidades» naturales y divinas, sino que permitían una vía de interpretación de esas dos instancias en una visión totalizadora en la que la Trinidad se presentaba como metáfora por excelencia. Por otro lado, el humanismo renacentista aliviado ya de la metáfora anterior, y entusiasmado con una nueva -el hombre- se aventuró a explorar y a inventar bordes y límites por los que transitara su renovada humanidad.

Learning by failure in entrepreneurship - Case study of selling Xylitol to Poland

My thesis aim is to find out what factors that can explain the entrepreneurial failure in my own entrepreneurial venture to sell Finnish xylitol product to Poland. In addition I seek to answer to the question of why Finnish xylitol products are not sold in Poland. This area has not been studied prior to my thesis. The study method is case study. Data collection is based on auto-ethnographic method where field notes and documents generated from my business venture were used. I discovered that Polish consumers are not exposed to consumer education of xylitol health benefits. The underlying potential in xylitol products expansion from Finland to foreign countries is still mostly unleveraged. My business venture failed because I was not able to convince Finnish confectionery companies Fazer and Cloetta to give enough support for my project. Polish dentists were also opposed having a competitor from Finland to Wrighley company which dominates the Polish confectionery markets.

Opening a Distillery: Legal Details That Producers Need to Know

Like any new business venture, starting a distillery requires careful planning and a major investment of resources. Marylanders interested in opening a distillery should consider taking classes and talking to existing distillers before starting out on their own. This guide is intended to provide an overview of the legal process of getting the required licenses and permits needed to open a distillery.

Ernest Mercier, le pétrole et la France

Ernest Mercier est l’un des patrons les plus influents de l'entre-deux-guerres en France. Ses différentes activités industrielles l'ont conduit à siéger sur de vastes pans de l’économie française, notamment du secteur énergétique. La thèse retrace la carrière pétrolière d'un homme qui a joué un rôle central pour le développement d'une industrie devenue stratégique, mais qui est embryonnaire lorsqu'il rejoint ce secteur après la Première Guerre mondiale. Mercier assiste et assure la création d'une industrie pétrolière nationale. Les obstacles se font légion contre les ambitions pétrolières de la France. Elle se présente bien tard sur un marché étroitement contrôlé par de puissants trusts. La recherche et l'exploitation pétrolière demandent d'importantes ressources, et aucune société française n'a les moyens d'une politique indépendante. Certaines banques se lancent alors dans les affaires de pétrole en s'alliant aux grands trusts internationaux. C'est le cas de Paribas; la gestion de ses avoirs roumains représente la première expérience de Mercier dans ce secteur. L'État s'intéresse aussi au pétrole, il devient un acteur incontournable. Le gouvernement français n'a pourtant pas les moyens de ses ambitions dans le domaine pétrolier. La politique nationale mise en place durant l'entre-deux-guerres doit faire appel à l'épargne privée française. La création d'une compagnie nationale, la Compagnie française des pétroles, en 1924 regroupe ainsi les différentes banques et sociétés intéressées au pétrole. Mercier est personnellement choisi par le président Raymond Poincaré pour mener à bien cette mission. Cette carrière s'articule donc autour d'un fragile équilibre entre milieux privés et gouvernement. Mercier devient rapidement l'intermédiaire incontournable qui régit ces relations. La thèse s'appuie sur les archives bancaires et industrielles, mais aussi sur celles du gouvernement français et de ses différents ministères. Cette analyse de la carrière d'Ernest Mercier permet de retracer les origines du secteur pétrolier français et l'action déterminante d'un homme. Elle expose les mécanismes d'influence d'une puissante banque d'affaires et les conflits d'intérêts qu'engendre l'exploitation pétrolière.

Processo biotecnológico voltado a produção de estoques poliploides do camarão-branco litopenaeus vannamei (decapoda, penaeidae)

without practical results so far. Protocols used in biotechnological cultured aquatic organisms aimed at increasing growth rates and disease resistance, have been studied and perfected. Among the available techniques, the application of chromosomal manipulation, although still nascent, is presented as a tool aimed at mitigating ecological and economical issues in shrimp farming. The polyploidization artificial method already employed in fish and shellfish, has been widely researched for use in farmed shrimp. Some limitations of this method of expansion in shrimp refer to a better knowledge of cytogenetic aspects, the level of sexual dimorphism and performance in growing conditions. To contribute on some of these issues, the present study aimed to characterize cytogenetic species Litopenaeus vannamei (Decapoda) and Artemia franciscana (Anostraca), analyze the effectiveness of methods for detection of ploidy, through the use of flow cytometry in processes of induction polyploidy cold thermal shock at different stages of development of newly fertilized eggs. Additionally, aimed also the qualitative and quantitative comparison of larval development between diploid and polyploid organisms, besides the identification of sexual dimorphism in L. vannamei, through geometric morphometrics. The results provide information relevant to the improvement and widespread use of biotechnological methods applied toward national productivity in shrimp farming

STRUCTURE AND FUNCTION OF THE GROUP III CHAPERONINS, A UNIQUE CLADE OF PROTEIN FOLDING NANOMACHINES

The survival and descent of cells is universally dependent on maintaining their proteins in a properly folded condition. It is widely accepted that the information for the folding of the nascent polypeptide chain into a native protein is encrypted in the amino acid sequence, and the Nobel Laureate Christian Anfinsen was the first to demonstrate that a protein could spontaneously refold after complete unfolding. However, it became clear that the observed folding rates for many proteins were much slower than rates estimated in vivo. This led to the recognition of required protein-protein interactions that promote proper folding. A unique group of proteins, the molecular chaperones, are responsible for maintaining protein homeostasis during normal growth as well as stress conditions. Chaperonins (CPNs) are ubiquitous and essential chaperones. They form ATP-dependent, hollow complexes that encapsulate polypeptides in two back-to-back stacked multisubunit rings, facilitating protein folding through highly cooperative allosteric articulation. CPNs are usually classified into Group I and Group II. Here, I report the characterization of a novel CPN belonging to a third Group, recently discovered in bacteria. Group III CPNs have close phylogenetic association to the Group II CPNs found in Archaea and Eukarya, and may be a relic of the Last Common Ancestor of the CPN family. The gene encoding the Group III CPN from Carboxydothermus hydrogenoformans and Candidatus Desulforudis audaxviator was cloned in E. coli and overexpressed in order to both characterize the protein and to demonstrate its ability to function as an ATPase chaperone. The opening and closing cycle of the Chy chaperonin was examined via site-directed mutations affecting the ATP binding site at R155. To relate the mutational analysis to the structure of the CPN, the crystal structure of both the AMP-PNP (an ATP analogue) and ADP bound forms were obtained in collaboration with Sun-Shin Cha in Seoul, South Korea. The ADP and ATP binding site substitutions resulted in frozen forms of the structures in open and closed conformations. From this, mutants were designed to validate hypotheses regarding key ATP interacting sites as well as important stabilizing interactions, and to observe the physical properties of the resulting complexes by calorimetry.

Reconfiguring or reproducing intra-professional boundaries? Specialist expertise, generalist knowledge and the ‘modernization’ of the medical workforce

Efforts to ‘modernize’ the clinical workforce of the English National Health Service have sought to reconfigure the responsibilities of professional groups in pursuit of more effective, joined-up service provision. Such efforts have met resistance from professions eager to protect their jurisdictions, deploying legitimacy claims familiar from the insights of the sociology of professions. Yet to date few studies of professional boundaries have grounded these insights in the specific context of policy challenges to the inter- and intra-professional division of labour, in relation the medical profession and other health-related occupations. In this paper we address this gap by considering the experience of newly instituted general practitioners (family physicians) with a special interest (GPSIs) in genetics, introduced to improve genetics knowledge and practice in primary care. Using qualitative data from four comparative case studies, we discuss how an established intra-professional division of labour within medicine—between clinical geneticists and GPs—was opened, negotiated and reclosed in these sites. We discuss the contrasting attitudes towards the nature of genetics knowledge and its application of GPSIs and geneticists, and how these were used to advance conflicting visions of what the nascent GPSI role should involve. In particular, we show how the claims to knowledge of geneticists and GPSIs interacted with wider policy pressures to produce a rather more conservative redistribution of power and responsibility across the intra-professional boundary than the rhetoric of modernization might suggest.

Small and medium enterprise competitiveness: Internationalization strategy

Field lab: SME competitiveness

Tecnologia social para autogestão : um estudo em empreendedorismo econômico solidário da cadeia produtiva da cajucultura no Rio Grande do Norte

The theme of this dissertation is social technology for self-management: a study in economic enterprise supportive of Rio Grande do Norte. The research aimed to obtain evidence that the reapplication of technology management, basic economic-financial and pricing, as production costs, has the potential to contribute to the self-management of APABV. The social technology and self-management are theoretical frameworks used and where workers are central figures in both the generation and replication of technologies that are compatible with their realities, as in the conduct and management approach adopted by them in their venture, they are makers decisions. To achieve the proposed objective was accomplished participatory research, which was used in addition to document analysis, participatory methodologies such as the construction of the DRP, group interview, experience in the production environment and family of entrepreneurs APABV. This research allowed the management technologies such as spreadsheets controls basic economic and financial costs, when socialized and understood by workers has the potential informational and become part of their daily decision-making process of the project, making up social technology